Opiates like morphine sulfate are the most effective analgesics for treating acute and chronic severe pain, but their use is limited by development of tolerance and hypersensitivity to innocuous and noxious stimuli. The mechanisms of such opiate-induced hyperalgesia and antinociceptive tolerance are unclear. Peroxynitrite (ONOO-) produced from superoxide (O2.-) and nitric oxide (.NO), is a potent pro-inflammatory and pro- apoptotic reactive nitrogen species now implicated in hyperalgesia. Our central HYPOTHESIS is that endogenous ONOO- is the proximal molecule in a cascade of signaling events leading to morphine-induced hyperalgesia and antinociceptive tolerance. In mice we showed that repeated morphine administration leads to nitrotyrosine (NT, a marker of ONOO-) formation. The role of ONOO- was proven by showing that co- administering morphine with inhibitors of .NO synthase, scavengers of O2.- or novel ONOO- decomposition catalysts attenuated NT formation and prevented antinociceptive tolerance. These associative and causal links involving ONOO- and antinociceptive tolerance coincided with: 1) post-translational nitration of MnSOD, a glutamate transporter (GLT-1), and glutamine synthase (GS);2) increased formation of pro-inflammatory cytokines;and 3) oxidative DNA damage and activation of the nuclear factor poly(ADP-ribose) polymerase. Inhibiting ONOO- attenuated these changes. Finally, results with systemic administration of ONOO- decomposition catalysts were confirmed by intrathechal dosing, thus supporting its role at the level of the spinal cord. Three Specific Aims will test our hypothesis. Specific Aim 1 will define the associative and causal link between spinal ONOO- and the development of morphine-induced hypersensitivity and antinociceptive tolerance. Specific Aim 2 will identify, using pharmacological and genetic approaches, the contribution of the NADPH oxidase as source of O2.- and thus, de novo ONOO- formation during the development of morphine hypersensitivity and antinociceptive tolerance. Specific Aim 3 will elucidate the molecular and biochemical mechanisms whereby ONOO- modulates hyperalgesia and antinociceptive tolerance. Results from these studies will define the important role of ONOO- in the development of antinociceptive tolerance providing the rationale towards the development of ONOO- decomposition catalysts as adjuncts to opiates for the management of chronic pain. Project Narrative: Chronic pain affects approximately 86 million Americans and cost at least $100 billion annually in medical expenses and reduced work productivity. PUBLIC HEALTH RELEVANCE Opioid drugs such as morphine are the most effective analgesics for treating severe chronic pain, but its pain-relieving action is often diminished during chronic administration, necessitating dose escalation that reduces quality of life for the patient. Our studies will determine for the first time that removal of peroxynitrite with novel agents, restores the pain-relieving action of morphine opening a new frontier in chronic pain management thereby improving the associated socioeconomic consequences.